Carotenoids are plant pigments available from the diet that may have important effects in the human body. For example, it is thought that many carotenoids seem to have positive effects on immune function, which can be critical in stopping cancer. Carotenoids also have an antioxidant effect which may protect against heart disease. Further, some studies have shown that carotenoids may help reduce the risk of degenerative diseases, such as age-related macular degeneration.
Recently, researchers have focused on the carotenoids alpha-carotene (in carrots), lycopene (in red fruits and vegetables, such as tomatoes and red peppers), beta cryptoxanthin (in oranges), and lutein and zeaxanthin (in broccoli and leafy green vegetables). Some research shows people with deficiencies of these substances are more likely to develop certain kinds of cancer, particularly lung cancer, supporting the theory that increased consumption of these carotenoids might protect against cancer and that identification of a deficiencies of these substances may alert an individual to possible health concerns. One study particularly looked at lutein, a carotenoid found in many vegetables and fruits, including spinach, broccoli, lettuce, tomatoes, oranges, carrots, celery, and greens. The study evaluated nearly 2,000 people with colon cancer and found that lutein intake in the study group was considerably lower than it was in people who were cancer-free.
It has been demonstrated that carotenoids offer some degree of biologic protection against the formation of malignancies in various tissues. For example, carotenoids have been shown in animal models to prevent carcinoma formation in tissues such as skin, salivary gland, mammary gland, liver, and colon. In addition, low levels of carotenoids and related substances such as retinoids have been assessed as high risk factors for malignant lesions. For example, having low levels of the carotenoid lycopene has been associated with prostate and cervical cancer; the carotenoids lutein, zeaxanthin, alpha-carotene, and beta-carotene with lung cancer; and beta-carotene with oral cancer. Therefore, quantitatively measuring the chemical concentrations of these carotenoids, retinoids and other related substances provides an indicator of the risk or presence of cancer.
Skin cancer is the most common cancer in the United States. Methods to provide detection of the levels of chemicals which are associated with skin related malignancies are of great assistance to physicians and medical personnel in the early diagnosis and treatment of skin cancer.
It has been theorized that carotenoids in the skin provide biologic protection from cutaneous malignancy. Prior methods used to detect the presence of chemicals associated with skin cancer have mainly been through the analysis of tissues obtained by biopsies or other invasive procedures. A standard method presently used for measuring carotenoids is through high-performance liquid chromatography (HPLC) techniques. Such techniques require removal of large amounts of tissue sample from the patient for subsequent analysis and processing, which typically takes at least twenty-four hours to complete. Thus, this technique is both invasive and slow and also expensive.
A noninvasive method for the measurement of carotenoid levels in the macular tissue of the eye is described in U.S. Pat. No. 5,873,831, in which levels of carotenoids and related substances are measured using Raman spectroscopy. Raman spectroscopy can identify the presence and concentration (provided proper calibration is performed) of certain chemical compounds. Nearly monochromatic light is incident upon the sample to be measured, and the inelastically scattered light, which is of a different wavelength than the incident light, is detected and measured. The wavelength shift between the incident and scattered light is known as the Raman shift, and the shift corresponds to an energy which is the “fingerprint” of the vibrational or rotational energy state of certain molecules. Typically, a molecule exhibits several characteristic Raman active vibrational or rotational energy states, and the measurement of the molecule's Raman spectrum thus provides a fingerprint of the molecule, i.e., it provides a molecule-specific series of spectrally sharp vibration or rotation peaks. The intensity of the Raman scattered light corresponds directly to the concentration of the molecule(s) of interest.
One difficulty associated with Raman spectroscopy is the very low signal intensity which is inherent to Raman scattered light. It is well known that the scattered light intensity scales with the wavelength raised to the fourth power. The weak Raman signal must be distinguished from Rayleigh scattered light, which is elastically scattered light of the same wavelength as the incident light and which constitutes a much greater fraction of the total scattered light. The Raman signal can be separated from Rayleigh scattered light through the use of filters, gratings, or other wavelength separation devices; however, this can have the effect of further weakening the measured Raman signal through the additional attenuation that can occur when the light passes through a wavelength separation device. In practice, the Raman scattered light is extremely difficult to detect. One might attempt to increase the Raman signal by increasing the intensity of the incident light on the tissue sample. Lasers have been used as a light source to increase light intensity, but this can cause burning or degradation of the sample.
In order to overcome some of these difficulties, a technique known as resonance Raman spectroscopy has been used, as described in U.S. Pat. No. 5,873,831, referenced hereinabove. Such a technique is also described in U.S. Pat. No. 4,832,483. In resonance Raman spectroscopy, the incident illumination utilized has a wavelength which corresponds to the resonance wavelength corresponding to electron energy transitions of the molecules of interest. This has the effect of strongly enhancing the Raman output signal without using a higher intensity input signal, thereby avoiding damage to the sample which can be caused by laser burning. Also, these resonance Raman signals have much higher intensity than off-resonance Raman signals, which are virtually invisible at power levels that will not damage human tissue. Therefore, in resonance Raman spectroscopy only those Raman signals which belong to the species of interest are obtained.
In the above referenced U.S. Pat. No. 5,873,831, the resonance Raman technique is used to measure the levels of the carotenoids lutein and zeaxanthin, two chemicals which are associated with healthy macular tissue of the human eye. The above referenced U.S. Pat. No. 4,832,483 uses resonance Raman spectroscopy to measure certain carotenoids in blood plasma, and suggests the use of the ratios of the intensities of the Raman spectral peaks as a method of indicating the presence of various malignancy diseases.
Yet another difficulty associated with Raman measurements is that the substances of interest in the skin not only scatter incident light, but can absorb and subsequently fluoresce with substantial intensity. This fluorescence often comprises a very strong, spectrally broad signal that tends to “drown out” or overwhelm the Raman spectral peaks, increasing the difficulty of identification and quantification of the substances.
Fluorescence spectroscopy is itself another technique that can be used to measure amounts of chemical compounds in biological tissue. For example, U.S. Pat. No. 5,697,373 discloses use of fluorescence and/or Raman spectroscopy to detect tissue abnormality in the cervix. The disadvantage of fluorescence measurements is that since many different molecules fluoresce in broad bands of wavelengths, such measurements cannot be used to identify conclusively the presence or concentration of a particular substance.
U.S. Pat. No. 6,205,354 shows a system that uses a laser to excite tissue such as an area of skin on the hand and measure carotenoid levels through an observation of the spectrum of the resulting scattered light. The faint but narrowband Raman peak must be distinguished from the broadband fluorescence background in the spectrum (typically over 100 times brighter). The system in U.S. Pat. No. 6,205,354 uses a technique that estimates the level of fluorescence (or background light) underneath the Raman peak by doing a curve fit to portions of the spectrum adjacent to either side of the expected Raman peak, but not part of the peak. The spectral resolution necessary for this analysis severely limits the amount of light that can be collected from the tissue with a standard spectrometer, which the system relies on. It requires that the excited tissue area to be small, just a fraction of a millimeter. Lasers are a good source of monochromatic light that can be focused to a small spot for the Raman excitation, but lasers with wavelengths in the needed blue-green spectral region are expensive and somewhat difficult to keep stable in environments with varying physical conditions. Moreover, estimating the level of background light beneath the Raman peak based on measurements of light to either side of the peak is susceptible to error that can vary with specific hardware variability.
It would therefore be a significant advance to provide an apparatus and method to measure chemical concentrations in tissue using Raman spectroscopy which has sufficient spectral measurement sensitivity to accommodate a relatively large tissue excitation area and which does not require the use of a laser or a baseline estimate of fluorescence levels beneath the Raman peak. Such an apparatus would make more widely available safe, noninvasive, rapid, accurate, and specific measurement of the levels of carotenoids and other similar chemical compounds that are present in varying degrees in biological tissues.